M. Ayuso

Gender-specific early postnatal catch-up growth after intrauterine growth retardation by food restriction in swine with obesity/leptin resistance

The effects of undernutrition during pregnancy on prenatal and postnatal development of the offspring were evaluated in sows with obesity/leptin resistance. Females were fed, from day 35 of pregnancy onwards, a diet fulfilling either 100% (group control, n=10) or 50% of the nutritional requirements (group underfed, n=10). In the control group, maternal body weight increased during pregnancy (P<0.05) while it decreased or remained steady in the underfed group. At days 75 and 100 of gestation, plasma triglycerides were lower but urea levels were higher in restricted than in control sows (P<0.05 for both). Assessment of the offspring indicated that the trunk diameter was always smaller in the restricted group (P<0.01 at day 50, P<0.005 at days 75 and 100 and P<0.0001 at birth) while head measurements were similar through pregnancy, although smaller in the restricted than in the control group at birth (P<0.05). Newborns from restricted sows were also lighter than offspring from control females (P<0.01) and had higher incidence of growth retardation (P<0.01). Afterwards, during lactation, early postnatal growth in restricted piglets was modulated by gender. At weaning, males from restricted sows were still lighter than their control counterparts (P<0.05), while females from control and underfed sows were similar. Thus, the current study indicates a gender-related differential effect in the growth patterns of the piglets, with females from restricted sows evidencing catch-up growth to neutralise prenatal retardation and reaching similar development than control counterparts.

Comparative Analysis of Muscle Transcriptome between Pig Genotypes Identifies Genes and Regulatory Mechanisms Associated to Growth, Fatness and Metabolism

Iberian ham production includes both purebred (IB) and Duroc-crossbred (IBxDU) Iberian pigs, which show important differences in meat quality and production traits, such as muscle growth and fatness. This experiment was conducted to investigate gene expression differences, transcriptional regulation and genetic polymorphisms that could be associated with the observed phenotypic differences between IB and IBxDU pigs. Nine IB and 10 IBxDU pigs were slaughtered at birth. Morphometric measures and blood samples were obtained and samples from Biceps femoris muscle were employed for compositional and transcriptome analysis by RNA-Seq technology. Phenotypic differences were evident at this early age, including greater body size and weight in IBxDU and greater Biceps femoris intramuscular fat and plasma cholesterol content in IB newborns. We detected 149 differentially expressed genes between IB and IBxDU neonates (p < 0.01 and Fold-Change > 1. 5). Several were related to adipose and muscle tissues development (DLK1, FGF21 or UBC). The functional interpretation of the transcriptomic differences revealed enrichment of functions and pathways related to lipid metabolism in IB and to cellular and muscle growth in IBxDU pigs. Protein catabolism, cholesterol biosynthesis and immune system were functions enriched in both genotypes. We identified transcription factors potentially affecting the observed gene expression differences. Some of them have known functions on adipogenesis (CEBPA, EGRs), lipid metabolism (PPARGC1B) and myogenesis (FOXOs, MEF2D, MYOD1), which suggest a key role in the meat quality differences existing between IB and IBxDU hams. We also identified several polymorphisms showing differential segregation between IB and IBxDU pigs. Among them, non-synonymous variants were detected in several transcription factors as PPARGC1B and TRIM63 genes, which could be associated to altered gene function. Taken together, these results provide information about candidate genes, metabolic pathways and genetic polymorphisms potentially involved in phenotypic differences between IB and IBxDU pigs associated to meat quality and production traits.

Prenatal programming in an obese swine model: sex-related effects of maternal energy restriction on morphology, metabolism and hypothalamic gene expression.

Maternal energy restriction during pregnancy predisposes to metabolic alterations in the offspring. The present study was designed to evaluate phenotypic and metabolic consequences following maternal undernutrition in an obese pig model and to define the potential role of hypothalamic gene expression in programming effects. Iberian sows were fed a control or a 50 % restricted diet for the last two-thirds of gestation. Newborns were assessed for body and organ weights, hormonal and metabolic status, and hypothalamic expression of genes implicated in energy homeostasis, glucocorticoid function and methylation. Weight and adiposity were measured in adult littermates. Newborns of the restricted sows were lighter (P <0·01), but brain growth was spared. The plasma concentration of TAG was lower in the restricted newborns than in the control newborns of both the sexes (P <0·01), while the concentration of cortisol was higher in females born to the restricted sows (P <0·04), reflecting a situation of metabolic stress by nutrient insufficiency. A lower hypothalamic expression of anorexigenic peptides (LEPR and POMC, P <0·01 and P <0·04, respectively) was observed in females born to the restricted sows, but no effect was observed in the males. The expression of HSD11B1 gene was down-regulated in the restricted animals (P <0·05), suggesting an adaptive mechanism for reducing the harmful effects of elevated concentrations of cortisol. At 4 and 7 months of age, the restricted females were heavier and fatter than the controls (P< 0·01). Maternal feed restriction induces asymmetrical growth retardation and metabolic alterations in the offspring. Differences in gene expression at birth and higher growth and adiposity in adulthood suggest a female-specific programming effect for a positive energy balance, possibly due to overexposure to endogenous stress-induced glucocorticoids.

Maternal malnutrition and offspring sex determine juvenile obesity and metabolic disorders in a swine model of leptin resistance.

The present study aimed to determine, in a swine model of leptin resistance, the effects of type and timing of maternal malnutrition on growth patterns, adiposity and metabolic features of the progeny when exposed to an obesogenic diet during their juvenile development and possible concomitant effects of the offspring sex. Thus, four groups were considered. A CONTROL group involved pigs born from sows fed with a diet fulfilling their daily maintenance requirements for pregnancy. The treated groups involved the progeny of females fed with the same diet but fulfilling either 160% or 50% of pregnancy requirements during the entire gestation (OVERFED and UNDERFED, respectively) or 100% of requirements until Day 35 of pregnancy and 50% of such amount from Day 36 onwards (LATE-UNDERFED). OVERFED and UNDERFED offspring were more prone to higher corpulence and fat deposition from early postnatal stages, during breast-feeding; adiposity increased significantly when exposed to obesogenic diets, especially in females. The effects of sex were even more remarkable in LATE-UNDERFED offspring, which had similar corpulence to CONTROL piglets; however, females showed a clear predisposition to obesity. Furthermore, the three groups of pigs with maternal malnutrition showed evidences of metabolic syndrome and, in the case of individuals born from OVERFED sows, even of insulin resistance and the prodrome of type-2 diabetes. These findings support the main role of early nutritional programming in the current rise of obesity and associated diseases in ethnics with leptin resistance.

Developmental Origins of Health and Disease in swine: implications for animal production and biomedical research

The concept of Developmental Origins of Health and Disease (DOHaD) addresses, from a large set of epidemiological evidences in human beings and translational studies in animal models, both the importance of genetic predisposition and the determinant role of maternal nutrition during pregnancy on adult morphomics and homeostasis. Compelling evidences suggest that both overnutrition and undernutrition may modify the intrauterine environment of the conceptus and may alter the expression of its genome and therefore its phenotype during prenatal and postnatal life. In fact, the DOHaD concept is an extreme shift in the vision of the factors conditioning adult phenotype and supposes a drastic change from a gene-centric perspective, only modified by lifestyle and nutritional strategies during juvenile development and adulthood, to a more holistic approach in which environmental, parental, and prenatal conditions are strongly determining postnatal development and homeostasis. The implications of DOHaD are profound in all the mammalian species and the present review summarizes current knowledge on causes and consequences of DOHaD in pigs, both for meat production and as a well-recognized model for biomedicine research.

Early-postnatal changes in adiposity and lipids profile by transgenerational developmental programming in swine with obesity/leptin resistance

Maternal malnutrition during pregnancy, both deficiency and excess, induces changes in the intrauterine environment and the metabolic status of the offspring, playing a key role in the growth, status of fitness/obesity and appearance of metabolic disorders during postnatal life. There is increasing evidence that these effects may not be only limited to the first generation of descendants, the offspring directly exposed to metabolic challenges, but to subsequent generations. This study evaluated, in a swine model of obesity/leptin resistance, the existence and extent of transgenerational developmental programming effects. Pre- and postnatal development, adiposity and metabolic features were assessed in the second generation of piglets, descendant of sows exposed to either undernutrition or overnutrition during pregnancy. The results indicated that these piglets exhibited early-postnatal increases in adiposity and disturbances in lipid profiles compatible with the early prodrome of metabolic syndrome, with liver tissue also displaying evidence of paediatric liver disease. These features indicative of early-life metabolic disorders were more evident in the males that were descended from overfed grandmothers and during the transition from milk to solid feeding. Thus, this study provides evidence supporting transgenerational developmental programming and supports the necessity for the development of strategies for avoiding the current epidemics of childhood overweight and obesity.

Developmental Stage, Muscle and Genetic Type Modify Muscle Transcriptome in Pigs: Effects on Gene Expression and Regulatory Factors Involved in Growth and Metabolism

Iberian pig production includes purebred (IB) and Duroc-crossbred (IBxDU) pigs, which show important differences in growth, fattening and tissue composition. This experiment was conducted to investigate the effects of genetic type and muscle (Longissimus dorsi (LD) vs Biceps femoris (BF)) on gene expression and transcriptional regulation at two developmental stages. Nine IB and 10 IBxDU piglets were slaughtered at birth, and seven IB and 10 IBxDU at four months of age (growing period). Carcass traits and LD intramuscular fat (IMF) content were measured. Muscle transcriptome was analyzed on LD samples with RNA-Seq technology. Carcasses were smaller in IB than in IBxDU neonates (p < 0.001), while growing IB pigs showed greater IMF content (p < 0.05). Gene expression was affected (p < 0.01 and Fold change > 1.5) by the developmental stage (5,812 genes), muscle type (135 genes), and genetic type (261 genes at birth and 113 at growth). Newborns transcriptome reflected a highly proliferative developmental stage, while older pigs showed upregulation of catabolic and muscle functioning processes. Regarding the genetic type effect, IBxDU newborns showed enrichment of gene pathways involved in muscle growth, in agreement with the higher prenatal growth observed in these pigs. However, IB growing pigs showed enrichment of pathways involved in protein deposition and cellular growth, supporting the compensatory gain experienced by IB pigs during this period. Moreover, newborn and growing IB pigs showed more active glucose and lipid metabolism than IBxDU pigs. Moreover, LD muscle seems to have more active muscular and cell growth, while BF points towards lipid metabolism and fat deposition. Several regulators controlling transcriptome changes in both genotypes were identified across muscles and ages (SIM1, PVALB, MEFs, TCF7L2 or FOXO1), being strong candidate genes to drive expression and thus, phenotypic differences between IB and IBxDU pigs. Many of the identified regulators were known to be involved in muscle and adipose tissues development, but others not previously associated with pig muscle growth were also identified, as PVALB, KLF1 or IRF2. The present study discloses potential molecular mechanisms underlying phenotypic differences observed between IB and IBxDU pigs and highlights candidate genes implicated in these molecular mechanisms.

Dietary vitamin A restriction affects adipocyte differentiation and fatty acid composition of intramuscular fat in Iberian pigs

The aim of this study was to investigate whether dietary vitamin A level is associated with differences in adipocyte differentiation or lipid accumulation in Iberian pigs at early growing (35.8kg live weight) and at finishing (158kg live weight). Iberian pigs of 16.3kg live weight were allocated to two feeding groups, one group received 10,000IU of vitamin A/kg diet (control); the other group received a diet with 0IU of vitamin A (var) for the whole experimental period. The dietary vitamin A level had no effect on growth performance and carcass traits. The early suppression of vitamin A increased the preadipocyte number in Longissimus thoracis (LT) muscle in the early growth period (P<0.001) and the neutral lipid content and composition (higher MUFA and lower SFA content) at the end of the finishing period (P<0.05). Vitamin A restriction in young pigs increases their lipogenic potential without affecting carcass traits.

Long term vitamin A restriction improves meat quality parameters and modifies gene expression in Iberian pigs.

Vitamin A is a key regulator of gene expression, influencing adipogenesis and lipid metabolism in animal tissues. This experiment was conducted to assess the effect of dietary vitamin A level and administration time on productive traits, intramuscular fat (IMF) content in ham muscles, tissue fatty acid composition, and expression of a panel of adipogenic and lipogenic candidate genes in Iberian pigs. Sixty piglets of 16.3 kg (SD = 2.5 kg) live weight (LW) were either fed a vitamin A-enriched diet (10,000 IU vitamin A/kg; CONTROL, n = 20) or a diet without supplemented vitamin A, applied from 16.3 kg (SD = 2.5 kg; early restriction group, ER, n = 20) or from an average weight of 35.8 kg (SD = 3.1 kg; late restriction group, LR, n = 20). Two slaughters were performed when pigs reached the averaged weights of 101.4 (SD = 4.1 kg) and 157.9 kg LW (SD = 7 kg) and samples from liver, heart, and backfat were obtained in both sacrifice times. In addition, ham subcutaneous fat and Semimembranosus (SM) and Biceps Femoris (BF) muscles were sampled at the last sacrifice. Dietary vitamin A level produced no effect on carcass traits in any of the harvests, while a small effect was observed on fatty acid composition in backfat at 101.4 kg LW. However, at 157.9 kg LW, the ER and LR groups showed higher MUFA content and lower SFA content in backfat, ham fat, and IMF (P < 0.01). In IMF, a decrease in n-6/n-3 PUFA ratio was observed in the restricted groups (P < 0.005). Intramuscular fat content in SM muscle was greater (P < 0.05) in the ER group than in the CONTROL and LR groups, while no difference was detected in BF muscle. Little effect of dietary vitamin A was observed in liver. Regarding changes in gene expression, ACSL4, CEBPB, and IGF1 genes were upregulated (P < 0.0001, P < 0.0001, and P < 0.05, respectively) in the ER group in hepatic tissue, whereas CRABPII and SCD genes were upregulated (P < 0.05) in the same group in adipose tissue. On the other hand, was downregulated ( < 0.05) in the ER group in adipose tissue. Results found in this experiment show that long-term restriction of dietary vitamin A has a positive effect on nutritional and sensorial parameters of ham meat. Moreover, gene expression results were consistent with the vitamin A transcriptional regulation of adipogenesis and lipogenesis and with the changes observed in meat and fat composition.

Fetal and early-postnatal developmental patterns of obese-genotype piglets exposed to prenatal programming by maternal over- and undernutrition.

The present study evaluated the effect of nutritional imbalances during pregnancy, either by excess or deficiency, on fertility and conceptus development in obese-genotype swine (Iberian pig). Twenty-five multiparous sows were fed, from mating to farrowing, with a standard diet fulfilling either 1.6 folds their daily maintenance requirements for pregnancy (overfed group, n = 12) or only the 50% of such requirements (underfed group, n = 13). Ten out of 12 overfed but only two out of 13 underfed sows became pregnant (P<0.005). Fetal development was determined in the pregnant females at Days 35, 50, 75 and 90 of pregnancy. The embryos from undernourished sows were smaller than the embryos from overfed females as early as at 35 days of pregnancy (P<0.05) and remained smaller until Day 90 of gestation. However, at the end of pregnancy, there were significant changes in the developmental patterns of fetuses. Thus, weight and size of the offspring from both nutritional treatments were finally similar at delivery; the same was found at weaning. There was thereafter a sex-related effect on the growth during the early-postnatal period, with male piglets of both nutritional origins being significantly heavier and more corpulent at weaning that their sisters (P<0.05). In conclusion, fetal growth conditioned by malnutrition from periconceptional stages is mainly regulated at the end of the pregnancy, so that ensure an adequate body-weight and size and, therefore, the survival of the offspring. Afterwards, the early-postnatal development of the offspring is affected by sex, independently from nutritional origin, with male piglets growing faster than females.